A salient feature of the septic shock state due to Gram negative bacteria is the presence of abnormalities of the coagulation mechanism, as manifested by disseminated intravascular coagulation, adrenal hemorrhage, thrombus formation and other well-known syndromes. Activation of coagulation in this setting is thought to result from the expression of tissue factor, a procoagulant cell surface cofactor, in the intravascular space by monocytes and endothelial cells. The pathogenesis of this host response to the Gram negative bacteria, especially the lipopolysaccharide (LPS), or endotoxin component of the cell wall, is thought to involve the elaboration of mediators, termed cytokines, such as tumor necrosis factor/cachectin (TNF) (1-3) and Interleukin 6, whose effects in target tissues result in many of the clinical findings characteristic of the septic shock state. However, the coagulopathy of Gram negative shock is not duplicated by any of the known endotoxin-derived host response mediators, leading us to identify alternate products which result in the induction of cellular tissue factor. Macrophages have been identified as the likely source of such products following exposure to endotoxin, a toxic component of the cell wall of Gram negative bacteria.
Macrophage mediators have a central role in the host response to multiple injurious stimuli. The study of endotoxin-elicited macrophage mediators, such as tumor necrosis factor (TNF), certain interleukins and macrophage inflammatory proteins, has emphasized the potential significance of these cytokines in the pathophysiology of the septic shock state. Abnormalities of the coagulation mechanism are a prominent feature of septic shock, and result, in large part, from the induction of tissue factor in cells in the intravascular space (especially monocytes and, probably, endothelial cells as well). Studies in which known cytokines have been infused into animals have not succeeded in reproducing the serious coagulopathy associated with Gram negative sepsis.
We have characterized a novel polypeptide (endotoxin-induced thrombosis factor) synthesized and secreted by macrophages (RAW 264.7) in response to endotoxin which induces tissue factor in monocytes and endothelium. Endotoxin-induced thrombosis factor has a role in the pathogenesis of shock and thrombosis, as a factor which can induce tumor necrosis (its activities in vitro suggest it will result in tumor necrosis in vivo), and in other clinically relevant situations (especially as a promoter of angiogenesis and white cell proliferation/development).
Septic shock is associated with profound abnormalities of the coagulation mechanism. Although known macrophage mediators, such as tumor necrosis factor (TNF.alpha.), reproduce certain aspects of the shock syndrome, they do not elicit the full range of coagulation abnormalities seen with lipopolysaccharide (LPS). The murine macrophage cell line RAW 264.7 was exposed to LPS and conditioned medium was subjected to sequential anion exchange chromatography, gel elution, and reversed phase (C2/C8) chromatography. The final product, which was homogeneous on SDS-PAGE, was a .apprxeq.55 kDa polypeptide (endotoxin-induced thrombosis factor) with a unique amino-terminal sequence, which induced tissue factor activity and antigen in cultured human ECs. The activity of endotoxin-induced thrombosis factor was destroyed by heat and treatment with trypsin. Half-maximal induction of tissue factor on ECs occurred at a concentration of .apprxeq.150 pM. In addition, the polypeptide was effective in inducing tissue factor activity in human monocytes. In conclusion, the new polypeptide is an LPS-elicited macrophage mediator which may contribute to the coagulopathy characteristic of Gram negative septicemia.
Transformed murine macrophages (RAW) secrete the novel polypeptide, endotoxin-induced thrombosis factor, whose expression is elicited after exposure to LPS and which potently induces tissue factor in human endothelial cells and monocytes. This factor is responsible for the majority of the endothelial cell tissue factor induced by LPS. Endotoxin-induced thrombosis factor has an important role in the pathogenesis of the coagulopathy associated with Gram negative sepsis. This invention is a novel polypeptide released by RAW cells stimulated by endotoxin. Inhibiting activity of endotoxin-induced thrombosis factor will prevent abnormal formation of blood clots in septic shock and other conditions.
Cytokines, specifically interleukin-1 and tumor necrosis factor, are induced by endotoxin and induce tissue factor in endothelial cells. Interleukin-6 is also induced by endotoxin, but does not induce tissue factor. However, the novel polypeptide, endotoxin-induced thrombosis factor, is an especially potent tissue factor inducer, and as such is responsible for the major amount of tissue factor induction in Gram negative sepsis.